Integrins are alphabeta3 heterodimeric cell surface receptors that mediate cell-cell and cell-matrix interactions in metazoa, thus contribute to fundamental cellular processes such as cell migration, proliferation and survival. Integrins are often expressed on the cell surface in an inactive state (unable to bind physiological ligands in solution), thereby preventing cells from inappropriately adhering to each other or to the extracellular matrix unless activated by a physiologic stimulus. Then, integrins undergo rapid and reversible changes in affinity and avidity. The recent crystal structure of the complete ectodomain of an integrin revealed an unexpected bent conformation where the ligand-binding head folds back at the "knees" to abut the lower legs. The earlier EM images showing genuextended integrins, led to the hypothesis that switching to high affinity necessitates a genuextension of the integrin, much like the opening of a jack-knife. Recent data indicate however that integrins can form stable complexes with physiologic ligands in solution in the bent state, indicating that the switch to high affinity occurs through alternative mechanisms. The structural basis of avidity modulation is also unknown. And despite a plethora of evidence supporting a role for kinases, phosphatases, proteases and small GTPases in affinity and avidity regulation, the precise pathways converging on the integrin remain to be defined. During the current funding period, we have determined the crystal structure of the integrin ectodomain alone and in complex with the prototypical Arg-Gly-Asp ligand and also determined the EM structure of a bent integrin in complex with a large physiologic ligand. We have also produced a new crystal of the integrin ectodomain, which appears to contain a multimeric form of the integrin in the asymmetric unit. Finally, an RNAi library targeting all human kinases, phosphatases, proteases and most oncogenes has been developed. This continuation will build on the above progress by 1) testing a new hypothesis for the structural basis of affinity regulation, 2) solving the structure of the multimeric integrin form, which may provide important insight into the structural basis of avidity regulation. 3) Identify novel intermediates that regulate inside-out activation of integrins using genetic (RNAi), immunochemical and biochemical approaches.